0
Fall
Investigation of Kanamycin Bacterial
Resistance Contamination on Chicken
Farms
Dominic A. Galanti
Partners: Anurag Garikipati, Kayla Conway, and Troy Merigliano
Pennsylvania State University
BIOL 110H
TA: Mark Goldy-Brown
Due: 12/2/13
Chicken Farm Lab Report
13
Chicken Farm Lab Report
Galanti1
Introduction
Throughout history scientists have developed theories about evolution. Such
evolution is evident from the commonly accepted theory of primates evolving into
humans. Humans along with most other organisms show effects of evolution, which
was used to help that species survive. Darwin’s Theory of Evolution is the basis for
this idea claiming that all living things go through the process of “natural selection”
where favorable genes are retained through future generations in the population
(O’Neil, 2013).
Throughout the ages diseases caused by bacteria have been a huge problem;
from the Spanish Flu of 1918 infecting a third or the world’s population and killing
nearly 40 million people (Billings, 2005) to contamination in food and water
systems lacking a proper vaccine. Specifically the bacteria E. coli, which has been
found to be a major disease in contaminated agricultural businesses resulting in
mass scares of disease in the public food system. Bacteria are not exempt from
Darwin’s idea of “natural selection.” Bacteria have been shown to develop resistance
to commercial drug antibiotics when exposed to these antibiotics for extended
periods of time. Mutations occur in the bacteria that decrease the effectiveness of
the antibiotic. These bacteria with the mutations live as opposed to the ones without
the mutation who die and this becomes an example of natural selection. Antibiotic
resistance is an obstacle in food production as many commercial agriculture
businesses use antibiotics to result in a higher profit margin (APUA, 2013). Once
these bacteria are released into the environment the bacteria can affect other farms,
wildlife, and humans who may be susceptible to the bacteria. Antibiotic resistant
Chicken Farm Lab Report
Galanti2
bacteria are harder to treat than common bacteria found in nature and once they
reproduce the genes for the antibiotic resistance can create more and harder to
treat bacterial diseases (APUA, 2013). Many strains of E. coli are harmless but some
have mutated and developed antibiotic resistance posing a major problem for
commercialized food (Perry 2012).
Antibiotic resistance is a particular problem on chicken farms where
chickens are kept in high populations and in close quarters, so the farmers
incorporate antibiotics into the feed to prevent disease. After using the same
antibiotics continuously a resistant strain of bacteria develops. Three bacterial
samples from separate chicken farms; Tucker’s Cluckers, Lucky’s Eggs, and Mia’s
Egg-cellent Eggs, were analyzed for antibiotic resistance of the drug kanamycin.
Kanamycin prevents bacterial growth by causing the mRNA to be translated
incorrectly (Cry, 2010).
An outbreak of gastroenteritis linked to these farms (Cry, 2010) is the cause
for this investigation in order to determine whether the outbreak is caused by a
shared source or by solitary factors, with contamination from one to three sources.
Percentage of bacterial contamination is to be determined to provide proper
remediation to the farms. If the bacteria on these farms contain the resistance to the
kanamycin on the same plasmid then the null hypothesis of the same source would
be correct otherwise alternate hypotheses could include two or three sources.
Methods
To determine contamination serial dilutions of 10-2, 10-4, and 10-6 are made
with micropippetors. These dilutions inoculate six petri dishes, three with nutrient
Chicken Farm Lab Report
Galanti3
agar and three with nutrient agar and kanamycin, through sterile technique. Sterile
beads are inserted into the petri dishes and gently, gravity rolled over the agar to
evenly distribute the dilutions. The same beads are used progressing from the
greatest dilution to the least but different beads are used in dishes including
kanamycin. The petri dishes must incubate for 24 hours then remain in cold storage
until the next step in the experiment.
After the inoculation bacteria from the kanamycin plates are added to tubes
containing the primers, labeled orange, yellow, and blue. This is done by touching a
white pippetor tip to a kanamycin resistant bacterial colony then dipping the tip
into the primer, a new tip is used for each primer. The orange, yellow and blue
primers are then run through PCR along with the control plasmids in primers red,
green, and pink.
Each petri dish is counted for bacterial colonies. Those dishes with a lawn,
characterized by bacteria in a dish which cannot be counted as individual colonies,
or those contaminated are considered N/A and unusable for determining the
kanamycin resistance frequency.
Create the agarose electrophoresis with agarose, 1XTAE buffer, and ethidium
bromide dye. Gloves are used in this step with the ethidium bromide, as it is a
mutagen (Cry, 2002). Buffer is added to the hardened gel until the buffer covers the
gel. Loading dye is added to each tube from the PCR and mixed thoroughly using a
new tip for each. A ladder is added to the agarose gel in the first well followed by the
six tubes of primers in wells 2-7. The gel electrophoresis is run with ~160 volts.
Chicken Farm Lab Report
Galanti4
When the tracking dye has reached halfway the voltage is stopped and gel removed
for a photograph under the UV light box for plasmid comparison. (Cry, 2010)
Results
Table 1: Results of Counted Bacteria from farm #3 with and without Kanamycin
10-2 Dilution
10-4 Dilution
10-6 Dilution
Group
1 Without
Lawn
497
9
Kayla
Kanamycin
Conway and With Kanamycin 1970
8
0
Troy
Frequency
of N/A
1.610%
0%
Merigliano
Kanamycin
Resistant
Bacteria
Group
2 Without
Lawn
Contaminated/ 72
Anurag
Kanamycin
~2240
Garikipati
With Kanamycin 323
2
0
and Dominic Frequency
of N/A
N/A
0
Galanti
Kanamycin
Resistant
Bacteria
In table 1 frequencies for the data were inconclusive as the frequencies were N/A or
0. In this model N/A represents calculations, which included a lawn or were
contaminated
Equation1: Kanamycin Resistance Frequency % = (Bacteria Colonies on
Kanamycin/Bacterial Colonies without Kanamycin) x 100
Table 2: Section Frequencies of Kanamycin Resistant Bacteria
Group Members
Farm
10-2
10-4
10-6
Average
Frequency Frequency Frequency Kanamycin
%
%
%
Resistance
Frequency
Emily Parchuke
Tucker's N/A
0.323
3.92
2.12%
Cluckers
Anu,Troy,Kayla,Dom Mia's
N/A
1.61
0
1.61
Eggcelent
Eggs
Laura, Angela, Sam,
Tucker's N/A
0.2219
0
0.2219
Shweta
Cluckers
Katie,Mikaela,Mary
Lucky
N/A
0.742
0
0.742
Elizabeth
Egg
Kaleb/Zico,Emily,
Lucky's
N/A
0.021
0.022
0.0215
Chicken Farm Lab Report
Khushboo
Diana, Ramya,
Victoria
Eggs
Mia's
Eggcelent
Eggs
Galanti5
N/A
0.0015
0
0.0015
Image 1: Gel Electrophoresis Picture under UV Light for Tucker’s Cluckers
Image 2: Gel Electrophoresis Picture under UV Light for Lucky’s Eggs
Image 3: Gel Electrophoresis Picture under UV Light for Mia’s Egg-cellent Eggs
Chicken Farm Lab Report
Galanti6
Figure 1:Gel Electrophoresis Image for Mia’s Egg-cellent Eggs
Well
1
Ladder 2KB
1.5KB
1KB
700BP
500BP
300BP 100BP
2
Orange
3
Blue
4
Yellow
X
5
Red
X
6
Green
X
7
Pink
X
Figure 1 shows the DNA fragment size of Mia’s Egg-cellent Eggs in the gel
electrophoresis image under the U.V. light.
Table 3: Gel Electrophoresis Results for Mia’s Egg-cellent Eggs
Well Number
Well Contents
Contents
1
Ladder
Molecular Weight
Standards
2
Orange
Plasmid DNA from
Colony 1
3
Blue
Plasmid DNA from
Colony 2
DNA Fragment Sizes
in Base Pairs
2KB, 1.5KB, 1KB,
700 BP, 500 BP, 300
BP, 100 BP
N/A
N/A
Chicken Farm Lab Report
Galanti7
4
Yellow
Plasmid DNA from
Colony 3
400
5
Red
Plasmid Standard A
600
6
Green
Plasmid Standard B
500
7
Pink
Plasmid Standard C
400
Table 3 shows the results from the gel electrophoresis and UV picture which links
Mia’s Egg-cellent Eggs to plasmid c. Any fragment sizes listed as N/A were
unidentifiable in the U.V. picture.
Table 4: Gene Location on Plasmids for Chicken Farms
Group
Farm
Plasmid 1
Plasmid 2
1
Plasmid 3
# of Base
Pairs
600 B.P.
1: Tuckers
X
Cluckers
2
2: Lucky’s
X
480 B.P.
Eggs
3
3: Mia’s
X
400 B.P.
Egg-cellent
Eggs
4
3: Mia’s
X
400 B.P.
Egg-cellent
Eggs
Table 4 links the genes for kanamycin resistance to their respective plasmid. The
results show that each farm’s contamination is the result of a separate plasmid.
Table 1 shows the data needed in order to calculate the frequency of
kanamycin resistant bacteria. Data shown as a lawn or contamination would give
inconclusive results for frequencies. Table 2 shows the data for all the farms with
Mia’s Egg-cellent Eggs having the lowest frequency and Tucker’s Cluckers has the
highest frequency. Images 1-3 show the gel electrophoresis results with the DNA
fragments ranging from 400-600 base pairs.
An obvious trend in the counting of the bacteria would be the decrease of
bacterial colonies in the kanamycin plates as this was evident in all of the groups’
Chicken Farm Lab Report
Galanti8
data. In addition each farm was linked or close to in base pair size as one of the
standard plasmids. There were no extraneous base pair sizes.
Discussion
The data supports the alternate hypothesis of three separate sources. The gel
electrophoresis is examined and the farm plasmids do not align with one another.
Each bacteria sample has a different result with Tucker’s Cluckers at 600 B.P.
matching plasmid 1, followed by Lucky’s Eggs at 480 B.P. matching plasmid 2, and
Mia’s Egg-cellent eggs at 400 B.P. matching plasmid 3. It can be concluded that the
contamination on these farms is from separate locations due to kanamycin
resistance on separate plasmids for each bacterial sample. It can also be concluded
that Tucker’s Cluckers farm was either contaminated first out of the farms or had
more ideal conditions for the bacteria to reproduce, as a higher frequency of
kanamycin resistant bacteria would conclude this.
Human error can result in the majority of the error in this experiment,
primarily with sterile technique. Contamination in inoculation of agar plates can
result from lack of flow hood or keeping the petri dish lid open to a non-sterile
environment. Additionally if pipet tips were not changed in dilutions or the
distribution beads were used from least to greatest dilution then the dilution of the
petri dishes would be incorrect and thus frequencies also incorrect.
Each farm had a kanamycin bacterial resistance of less than 5%. Remediation
for the three farms is the same as they all fall under 5%. It is recommended to send
all eggs to a pasteurization facility until the kanamycin resistance is under 1% for 8
weeks. The farms should monitor the contamination levels weekly and identify
Chicken Farm Lab Report
Galanti9
where the source of contamination is. In addition the antibiotics delivered to the
chickens should change in order to eliminate the contamination.
Future research of each farm could identify what the source of contamination
is. This requires a much larger sample size than this experiment. Experimentation of
kanamycin resistance in other agricultural settings would help to better understand
which plasmid is responsible for the antibiotic resistance for kanamycin and would
improve our understanding of antibiotic resistance overall. Studying how the
chicken eggs contract the disease could give us a better understanding of how
humans contract antibiotic resistant bacterial infections and what can be done to
prevent or treat these problems.
In conclusion the contamination on the three farms; Tucker’s Cluckers,
Lucky’s Eggs, and Mia’s Egg-cellent Eggs, has been caused by three separate sources
of contamination. Tucker’s Cluckers has either been infected longer or the
environment of Tucker’s Cluckers farm is more suitable for the kanamycin bacteria
to thrive. All three farms have contamination levels under 5%, therefore the
remediation procedures are identical; the source of contamination must be
identified and all eggs must be sent to a pasteurization facility until the
contamination has been under 1% for 8 weeks.
Works Cited
Alliance for Prudent Use of Antibiotics (2013). Science of Resistance: Antibiotics in
Agriculture. Retrieved from
http://www.tufts.edu/med/apua/about_issue/antibiotic_agri.shtml
Billings, Molly (2005). The Influenza Pandemic of 1918. Retrieved from
http://virus.stanford.edu/uda/
Chicken Farm Lab Report
Galanti10
Cyr, R., Hass C., Woodward D., and Ward A., 2010. Using Genes for Antibiotic
Resistance to Trace Source(s) of Bacterial Contamination. In, Biology 110: Basic
concepts and biodiverity course website. Department of Biology, The Pennsylvania
State University. http://www.bio.psu.edu/
Cyr, R., 2002. Title of the tutorial being cited. In, Biology 110: Basic concepts and
biodiverity course website. Department of Biology, The Pennsylvania State
University. http://www.bio.psu.edu/
O’ Neill, Dennis (2013). Darwin and Natural Selection. Retrieved from
http://anthro.palomar.edu/evolve/evolve_2.htm
Perry, Ann (2012). Sources of E. coli Are Not Always What They Seem. Retrieved
from http://www.ars.usda.gov/is/pr/2012/121129.htm